Prostate cancer is the fastest growing neoplasm in men with an estimated 244,000 new cases in the United States being diagnosed in 1995, of which approximately 44,000 deaths will result. Hormonal ablation therapy, either surgically or chemically with anti-androgens, is the main stay of treatment for advanced carcinoma of the prostate. However, androgen ablation therapy failed within 12-18 months with the disease becoming androgen independent. Following the failure of androgen therapy, the median patient survival time is eight months. Other approaches to treating prostate cancer--external radiation, radioactive seed therapy, cryotherapy, etc.--are directed toward organ confined disease of the prostate and are unable to treat metastatic tumors.
The prostate-specific antigen (PSA), a member of the human kallikrein gene family, is a Mr=34,000 chymotrypsin like protein that is synthesized exclusively by normal, hyperplastic, and malignant prostatic epithelia. Hence, the PSA's tissue-specific relationship has made it an excellent biomarker for identifying benign prostatic hyperplasia (BPH) and prostatic carcinoma (CaP), hereinafter CaP. Normal serum levels of PSA in blood are typically below 5 ng/ml, with elevated levels indicative of BPH or CaP. Serum levels of 200 ng/ml have been measured in end-stage metastatic CaP.
Another member of the kallikrein gene family, human glandular kallikrein-1 (hGK-1 or hKLK2, encoding the hK2 protein), shares a number of characteristics with PSA. First, both are expressed exclusively in the prostate and are up-regulated by androgens primarily by transcriptional activation. Wolf et al. (1992) Molec. Endocrinol. 6:753-762. Morris (1989) Clin. Exp. Pharm. Physiol. 16:345-351; Qui et al. (1990) J Urol. 144:1550-1556; Young etal. (1992) Biochem. 31:818-824. Second, hKLK2 and PSA mRNAs are synthesized and co-localize only in prostatic epithelia. Third, hKLK2 and PSA exhibit a high degree of amino acid sequence identity. Schedlich et al. (1987) DNA 6:429-437. Fourth, they have similar regulatory elements. There is approximately 80% nucleotide sequence identity between PSA and hKLK2 in the 5'-flanking region from -300 to -1 relative to the transcription initiation site. Young et al. (1992) Biochem. 31:818-824. Each promoter contains an androgen responsive element (ARE); their respective ARE's differ from one another by only 1 nucleotide. Schedlich et al. (1987) DNA 6:429-437; Murtha et al. (1993) Biochem. 32:6459-6464.
The levels of hK2 found in various tumors and in the serum of patients with prostate cancer differ substantially from those of PSA. Circulating hK2 in different relative proportions to PSA has been detected in the serum of patients with prostate cancer. Charlesworth et al. (1997) Urology 49:487-493. Expression of hK2 has been detected in each of 257 radical prostatectomy specimens analyzed. Darson et al. (1997) Urology 49:857-862. The intensity and extent of hK2 expression, detected using specific antibodies, increased from benign epithelium to high-grade prostatic intraepithelial neoplasia (PIN) and adenocarcinoma, whereas PSA and prostate acid phosphatase (PAP) displayed an inverse pattern of immunoreactivity. Darson et al. (1997) Urology 49:857-862. Indeed, it has been reported that a certain percentage of PSA-negative tumors have detectable hK2. Tremblay et al. (1997) Am. J Pathol. 150:455-459.
As mentioned above, both PSA and hKLK2 genes are up-regulated by androgens primarily by transcriptional activation. Androgen induction of gene expression requires the presence of an androgen receptor (AR). Typically, an androgen diffuses passively into the cell where it binds AR. The androgen-activated AR binds to specific DNA sequences called androgen-responsive elements (AREs or ARE sites). Once anchored to an ARE, the AR is able to regulate transcriptional activity in either a positive or negative fashion. Lindzey et al. (1994) Vitamins and Hormones 49: 383-432.
The AR belongs to a nuclear receptor superfamily whose members are believed to function primarily as transcription factors that regulate gene activity through binding to specific DNA sequences, hormone-responsive elements. CarsonJurica et al. (1990) Endocr. Rev. 11: 201-220. This family includes the other steroid hormone receptors as well as the thyroid hormone, the retinoic acid and the vitamin D.sub.3 receptors. The progesterone and glucocorticoid receptor are structurally most closely related to the AR. Tilley et al. (1989) Proc. Natl. Acad Sci. USA 86: 327331; Zhou et al. (1994) Recent Prog. Horm. Res. 49: 249-274; and Lindzey et al. (1994) Vit. Horm. 49: 383-432.
The AR gene itself is a target of androgenic regulation. In the prostate cancer cells lines PC3 and DU145, which do not express an endogenous AR, androgenic up-regulation of AR cDNA expression occurred in the transfected cells. Dai et al. (1996). Androgenic up-regulation of AR mRNA and protein was observed in PC3 cells that were stably transfected with the AR CDNA, suggesting that AR mRNA regulation also occurs when the CDNA is organized into chromatin. Dai et al. (1996).
The characterization of genes whose expression is limited to the prostate allows the development of screening methods which can identify substances capable of specifically altering the expression of prostate-specific genes.
In the last few years, numerous techniques have been developed for producing vast arrays of potential drug-like compounds. These compounds include not only oligomers, such as oligopeptides and oligonucleotides, but also synthetic organic compounds based on various core structures. In addition, various natural sources have been screened for active compounds, such as those found in jungles, the ocean and the like. Thus, there is a great proliferation of available compounds for screening for physiological activity.
The process of identifying prospective compounds having therapeutic activity is primarily held back by the absence of useful screening assays. In order for a screening assay to be useful, it should be capable of automation, allow for the screening of large numbers of samples without requiring extensive equipment or housing, be relatively inexpensive, and provide for a clear indication of activity. There is, therefore, substantial interest in identifying new screening assays which would allow for the screening of compounds which may have therapeutic activity in relation to prostate cancer.